The present invention relates to gene-targeted, non-human mammals comprising a human mutation in the non-human mammalian presenilin 1 (PS-1) FAD gene, methods of identifying compounds for treating Alzheimer""s disease, and to methods of treating Alzheimer""s disease.
Alzheimer""s disease (AD) is an age-dependent neurodegenerative disorder that leads to profound behavioral changes and dementia. Hallmark pathologies include the atrophy of brain gray matter as a result of the massive loss of neurons and synapses, and protein deposition in the form of both intraneuronal neurofibrillary tangles and extracellular amyloid plaques within the brain parenchyma. In addition, affected areas of the AD brain exhibit a reactive gliosis that appears to be a response to brain injury. Surviving neurons from vulnerable populations in AD show signs of metabolic compromise as indicated by alterations in the cytoskeleton (Wang et al., Nature Med., 1996, 2, 871-875), Golgi complex (Salehi et al., J. Neuropath. Exp. Neurol., 1995, 54, 704-709) and the endosomal-lysosomal system (Cataldo et al., Neuron, 1995, 14, 671-680).
Approximately 10 to 30% of AD cases are inherited in an autosomal dominant fashion and are referred to as xe2x80x9cfamilial Alzheimer""s diseasexe2x80x9d or xe2x80x9cFAD.xe2x80x9d Genetic linkage studies have revealed that FAD is heterogeneous and a majority of the cases have been linked to gene mutations on chromosomes 1, 14, 19, or 21 (reviewed in Siman and Scott, Curr. Opin. Biotech., 1996, 7, 601-607). Importantly, these individuals have been shown to develop the classical symptomatic and pathological profiles of the disease confirming that the mutations are associated with the development of the disease rather than a related syndrome. The locus on chromosome 14 is associated with a significant fraction of FAD, and mutations at the locus have been mapped to a single-copy gene, termed xe2x80x9cS182xe2x80x9d or xe2x80x9cpresenilin 1xe2x80x9d (PS-1), that encodes a 467 amino acid protein (Sherrington et al., Nature, 1995, 375, 754-760; Clark et al., Nature Genet., 1995, 11, 219-222). A closely related gene, xe2x80x9cSTM2xe2x80x9d or xe2x80x9cpresenilin 2xe2x80x9d (PS-2), located on chromosome 1, has been linked to two additional FAD kindreds including the descendants of German families from the Volga valley of Russia (Levy-Lahad et al., Science, 1995, 269, 973-977; Rogaev et al., Nature, 1995, 376, 775-778). PS-1 and PS-2 share an overall 67% amino acid sequence homology, and primary structure analysis indicates they are integral membrane proteins with 6 to 8 trans-membrane domains (Slunt et al., Amyloidxe2x80x94Int. J Exp. Clin. Invest., 1995, 2, 188-190; Doan et al., Neuron, 1996, 17, 1023-1030). Much of the information on function of the presenilins stems from the identification of a presenilin homolog in C. elegans termed xe2x80x9cSEL-12,xe2x80x9d a 6 to 8 trans-membrane protein that appears to participate in an intracellular signaling pathway mediated by the lin-12/glp-1/Notch family (Levitan and Greenwald, Nature, 1995, 377, 351-354). PS-1 and SEL-12 proteins share a 49% sequence homology and have similar membrane orientations. Importantly, both human PS-1 and PS-2 can rescue the mutant sel-12 phenotype in C. elegans, indicating a role for the presenilins in Notch signaling (Levitan et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 14940-14944).
FAD linked to the presenilins is highly penetrant and the aggressive nature of the disease suggests that the mutant protein participates in a seminal pathway of AD pathology. To date, over seventy FAD mutations have been identified in PS-1, and three FAD mutations have been found in PS-2. Most of the FAD mutations occur in conserved positions between the two presenilin proteins, suggesting that they are affecting functionally or structurally important amino acid residues. Interestingly, many of the mutated amino acids are also conserved in SEL-12. All but two of the presenilin mutations are missense mutations. One exception results in an aberrant RNA splicing event that climninates exon 9, creating an internally-deleted mutant protein (Perez-Tur et al., NeuroReport, 1995, 7, 297-301; Sato et al., Hum. Mutat. Suppl., 1998, 1, S91-94; and Prihar et al., Nature Med., 1999, 5, 1090). The other results in two deletion transcripts (xcex944 and xcex944cryptic) and one full-length transcript with the amino acid Thr inserted between 113 and 114 (DeJonghe et al., Hum. Molec. Genet., 1999, 8, 1529-1540). The latter transcript leads to the AD pathophysiology. These latter points, along with the genetic dominance of the disease, argue that disease pathogenesis in the presenilin kindreds requires the production of a mutant presenilin protein having a novel and detrimental function, rather than the simple loss or reduction of normal presenilin levels. The mutations do appear to disrupt normal presenilin function however, because mutant presenilins are not able to rescue or fully rescue the sel-12 phenotype (Levitan et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 14940-14944).
Expression profiles of the presenilins have been examined at a gross level but, so far, these analyses have yielded little information on the mechanism of disease pathogenesis. Both presenilin 1 and 2 are widely expressed in the CNS and peripheral tissues. In brain, expression is enriched in neurons but is apparent in both AD-vulnerable and resistant areas. Cellular localization studies indicate that the proteins accumulate primarily in the Golgi complex and endoplasmic reticulum but no significant alterations in expression levels or subcellular distribution have been attributed to the FAD mutations (Kovacs et al., Nature Med., 1996, 2, 224-229).
The presenilin proteins are processed proteolytically through two intracellular pathways. Under normal conditions, accumulation of 30 kD N-terminal and 20 kD C-terminal proteolytic fragments occurs in the absence of the full-length protein. This processing pathway is highly regulated and appears to be relatively slow, accounting for turnover of only a minor fraction of the full-length protein. The remaining fraction appears to be rapidly degraded in a second pathway by the proteasome (Thinakaran et al., Neuron, 1996, 17, 181-190; Kim et al., J. Biol. Chem., 1997, 272, 11006-11010). Proteolytic metabolism of PS-1 variants linked to FAD appears to be different, but the relevance of the change to pathogenesis is not known (Lee, et al., Nature Med., 1997, 3, 756-760).
One pathogenic role for the mutant presenilins in FAD appears to be related to effects on processing of the amnyloid precursor protein (APP) and production of the Axcex2 peptide, the primary proteinaceous component of the extracellular neuritic plaque in the AD brain. Elevated serum levels of the longer form of Axcex2 (Axcex242), considered to be the more pathogenic species of the Axcex2 peptides, have been measured in patients bearing PS-1 and PS-2 mutations (Scheuner et al., Nature Med., 1996, 2, 864-870). Additionally, FAD brains with PS-1 mutations have large amounts of Axcex2 deposition (Lemere et al., Nature Med., 1996, 2, 1146-1150; Mann et al., Ann. Neurol., 1996, 40, 149-156; Gxc3x3mez-Isla et al., Ann. Neurol., 1997, 41, 809-813). Elevated levels of Axcex21-42 were also found in cells transfected with mutant PS-1 or PS-2 and in mice expressing mutant PS-1 (Borchelt et al., Neuron, 1996, 17, 1005-1013; Duff et al., Nature, 1996, 383, 710-713; Citron et al., Nature Med., 1997, 3, 67-72; Murayama et al., Prog. Neuro-Psychopharmacol. Biol. Psychiatr., 1999, 23, 905-913; Murayama et al., Neurosci. Lett., 1999, 265, 61-63; Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581). The mechanism by which the mutant presenilins affect APP processing is not known, but these results do support a causative role of increased Axcex242 production in the development of FAD. Importantly, it is possible that mutant presenilins influence other AD pathogenic processes as well, such as presumptive intracellular signaling and cell death pathways involved directly or indirectly in neuronal dysfunction and degeneration.
Genetically-engineered animals have been used extensively to examine the function of specific gene products in vivo and their role in the development of disease phenotypes. The genetic engineering of mice can be accomplished according to at least two distinct approaches: (1) a transgenic approach where an exogenous gene is randomly inserted into the host genome, and (2) a gene-targeting approach where a specific endogenous DNA sequence or gene is partially or completely removed, or replaced by homologous recombination. The transgene of a transgenic organism is comprised generally of a DNA sequence encoding the protein sequence and a promoter that directs expression of the protein coding sequences. A transgenic organism expresses the transgene in addition to all normally-expressed native genes. The targeted gene of a gene-targeted animal, on the other hand, can be modified in one of two ways: (1) a functional form where a modified version of the targeted gene is expressed, or (2) a non-functional or xe2x80x9cnullxe2x80x9d form where the targeted gene has been disrupted resulting in loss or reduction of expression. If the targeted gene is a single copy gene and the animal is homozygous at the targeted locus, then, depending on the type of modification, the animal either does not express the targeted gene or expresses only a modified version of the targeted gene in absence of the normal form.
Transgenic mice expressing native and mutant forms of the presenilin proteins have been described (Borchelt et al., Neuron, 1996, 17, 1005-1013; Duff et al., Nature, 1996, 383, 710-713; Borchelt et al., Neuron, 1997, 19, 939-945; Citron et al., Nature Med., 1997, 3, 67-72; Chui et al., Nature Med., 1999, 5, 560-564; and Nakano et al., Eur. J. Neurosci., 1999, 11, 2577-2581). Although mice bearing mutations in PS-1 had elevated levels of Axcex21-42, they have not formed Axcex2 deposits characteristic of AD or shown behavioral deficits associated with AD. Neuronal loss has been described by one group (Chui et al., Nature Med., 1999, 5, 560-564). When transgenic mice with PS-1 mutations were crossed with transgenic mice bearing the Swedish APP mutations, there was marked acceleration in the formation of Axcex2 deposits (Borchelt et al., Neuron, 1997, 19, 939-945; Holcomb et al., Nature Med., 1998, 4, 97-100; Lamb et al., Nature Neurosci., 1999, 2, 695-697). Gene-targeted PS-1 null mice lacking one or both functional alleles of the PS-1 gene have also been described (Wong et al., Nature, 1997, 387, 288-292, and Shen et al., Cell, 1997, 89, 629-639). Mice in which both PS-1 alleles have been disrupted resulting in the complete loss of PS-1 expression are not viable and die shortly after birth. No abnormal phenotypes or changes in APP processing have been reported in mice lacking only one of the two PS-1 alleles, but inhibition of APP processing is found in neurons derived from PS-1 null mice (DeStrooper et al., Nature, 1998, 391, 387-390).
In the present application, a gene-targeting approach (Reaume et al., J. Biol. Chem., 1996, 271, 23380-23388, which is incorporated herein by reference in its entirety) generating AD models is described. One model employs the Swedish FAD mutation and xe2x80x9chumanizedxe2x80x9d mouse Axcex2 sequence in the APP gene (U.S. Pat. No. 5,777,194, which is incorporated herein by reference in its entirety). This mouse (APPNLh/NLh) produced normal levels of APP, overproduced human Axcex21-40 and 1-42, but did not deposit Axcex2 (Reaume et al., J. Biol. Chem., 1996, 271, 23380-23388). A human PS-1 mutation, the P264L mutation in particular, was introduced into the mouse PS-1 gene. The P264L mutation is a non-conservative amino acid substitution in the cluster of mutations in exon 8, causing an onset of FAD in the middle forties to middle fifties (Campion et al., Hum. Molec. Genet., 1995, 4, 2373-2377; Wasco et al., Nature Med., 1995, 1, 848). Crosses produced APPNLh/NLhxc3x97PS-1P264L/P264L double gene-targeted mice. These mice had elevated levels of Axcex21-42, sufficient to cause Axcex2 deposition. Mice bearing the PS-1P264L mutation were also crossed with Tg2576 mice that overexpress Swedish APP695 (Hsiao et al., Science, 1996, 274, 99-102; available from the Mayo Clinic, Rochester, Minn.). One distinct advantage of the present invention is that for heterozygous and homozygous gene-targeted mice, the fidelity of expression patterns of proteins is maintained since the expression is under the endogenous promoter. Further, expression levels of the holoprotein are not changed.
The present invention relates to a gene-targeted, non-human mammal comprising a gene encoding a mutant protein product of a mutated FAD presenilin-1 (PS-1) gene, a human FAD Swedish mutation, and a humanized Axcex2 mutation, and generational offspring thereof. The present invention also relates to a gene-targeted, non-human mammal comprising a gene encoding a mutant protein product of a mutated FAD presenilin-1 (PS-1) gene and a human Swedish APP695 mutation, and generational offspring thereof Preferably, the PS-1 gene has been mutated to contain the human P264L mutation (Wasco et al., Nature Medicine, 1995, 1, 848). In particular, the present invention relates to a mouse wherein a part of a mouse presenilin 1 gene encoding presenilin 1 protein has been replaced with a DNA sequence that results in a mouse presenilin 1 gene that contains a human mutation, most preferably a P264L mutation. Still more specifically, the base sequence of codon 264 of the mouse presenilin 1 gene is altered from CCG to CTT, which is the base sequence found to constitute the P264L mutation of humans. The mutated gene codon encodes leucine in place of proline in amino acid number 264 of presenilin 1. Additionally, and still more specifically, a nucleotide base in codon 265 of the mouse presenilin 1 gene is altered from adenosine to guanosine, but this change does not result in an amino acid change in the expressed protein. However, the combined sequence of codons 264 and 265, after the incorporation of the most preferred changes described above, results in a restriction enzyme site for the restriction enzyme AflII.
Accordingly, in one embodiment, the present invention features a non-human mammal and generational offspring homozygous for a targeted mutant PS-1 gene comprising a mutated FAD gene preferably a mouse presenilin 1 protein-encoding sequence comprising a human mutation, most preferably a P264L mutation, in place of the native presenilin 1 protein-encoding sequence. In another embodiment, the invention features a non-human mammal and generational offspring heterozygous for a targeted PS-1 gene comprising a mutated mouse FAD gene, preferably a mouse presenilin 1 protein-encoding sequence containing a human mutation, most preferably a P264L mutation, in place of the native presenilin 1 protein-encoding sequence.
The present invention is also directed to methods for identifying a compound for treating Alzheimer""s disease comprising administering a compound to a mammal heterozygous or homozygous for a mutation of the PS-1 gene and a human Swedish APP695 mutation, or generational offspring thereof, or to a mammal heterozygous or homozygous for a mutation of the PS-1 gene, a human FAD Swedish mutation, and a humanized Axcex2 mutation, and generational offspring thereof, and measuring the amount of Axcex242 peptide in a tissue sample from the mammal.
The present invention is also directed to methods of treating an individual suspected of having Alzheimer""s disease comprising administering to the individual an effective Alzheimer""s disease treatment amount of a compound identified by the method described above.
The present invention is also directed to compounds identified by any of the methods described above.